The Role Of Kupffer Cells In Oval Cell Response And Liver Protection During Liver Regeneration After 2-AAF/PH In Rats

Objectives:To assess the role of liposome encapsuled clodronate (Lip-Clod) depleting rat liver Kupffer cells, and to determine the regeneration time course of Kupffer cells after depletion.Methods:Rats were randomly divided into depleted group and control group. Lip-Clod was administered at a dosage of 0.1 ml/10g bodyweight intravenously. Control rats received the same volume of 0.9% NaCl. Two days after injection, RT-PCR was performed to detect mRNA level of Kupffer Cell Receptor (KCR). To assess phagocytosis, rats administered with or without Lip-Clod were injected with Indian ink and sacrificed 30 minutes later. Immunohistochemistry for ED1 and ED2 was performed to assess the fact of Kupffer cell depletion. Rat livers were harvested at several time points after the injection, and Kupffer cell regeneration was determined.Results:Two days after treatment of Lip-Clod, ED1 and ED2 positive cells were nearly absolutely disappeared. Meanwhile, expression of KCR mRNA could not be detected, and the dense carbon particle uptake was absent after Lip-Clod injection. Eight days after treatment, number of ED1 positive cells was significantly increased and returned to normal level at day 11. A small number of ED2 positive cells could be observed 11 days after treatment. There were only (4.8±1.7)cells in Kupffer cell depleted group compared to (17.7±2.1) in control group (P<0.01).Conclusions:Intravenous administration of a single dose of Lip-Clod can efficiently deplete liver Kupffer cells in two days, and the depletion lasts for at least one week.Objectives:Kupffer cells are known to play an important role in liver regeneration by secreting inflammatory mediators. In several oval cell expansion models, numerous Kupffer cells were observed mainly assembling in the periportal area, implying a potential role in oval cell mediated liver regeneration. In the present study, we investigated the role of Kupffer cells in oval cell response in 2-acetylaminofluorene/partial hepatectomy (2-AAF/PH) rat liver.Methods:All rats received treatment of 2-AAF/PH, and then randomly divided into Kupffer cell depleted group and non-depleted group. Kupffer cells were selectively depleted by injecting liposome encapsulated clodronate 48 hours before hepatectomy in depleted group rats, while control rats received the same dosage of normal saline. Liver samples were collected at several time points after PH. Liver weight/body weight were calculated to assess the extent of remnant liver regeneration. Immunohistochemistry for EpCAM, PCK and PCNA were performed. And Extent of oval cell expansion was evaluated by counting the positive cells as well as by measuring the basophilic ductular reaction areas in periportal region. Oval cell proliferative activity was determined by double immunofluorescent staining for EpCAM and PCNA, and apoptosis was determined by caspase-3 immunostaing and TUNEL staining. Real-time quantitative PCR was performed to detect mRNA levels of albumin and alpha fetoprotein. Kupffer cells were isolated from 2-AAF/PH rat livers, and non-serum medium suspension was collected. Concentration of tumor necrosis factor alpha (TNF-a) and interleukin 6 (IL-6) in both liver homogenates and cultured medium suspension was measured by enzyme-linked immunosorbent assay (ELISA). Expression of phosphor-STAT3 and total STAT3 were detected by western blot. Association between hepatic stellate cell and oval cell was detected by double immunofluorescent staining for EpCAM and alpha-smooth muscle actin.Results:Oval cell expansion was inhibited in the Kupffer cell depleted rats, as evidenced by decreased liver weight regain, periportal basophilic area of ductular reaction, and number of oval cells. Meanwhile, oval cell differentiation was also attenuated as a result of Kupffer cell depletion. In control rats, large foci of small basophilic hepatocytes could be observed by day 9 after hepatectomy, whereas they were nearly absent in Kupffer cell depleted rats accompanied with lower expression of albumin and higher expression of alpha fetoprotein at the mRNA level. However, the percentage of EpCAM positive oval cells labeled with PCNA was similar in both group, and we did not observe any difference in the number of apoptotic periportal OCs between the two groups. Kupffer cell-depletion decreased hepatic expression of TNF-a and IL-6, as well as phosphorylation of STAT3 during the early period following hepatectomy. Meanwhile, Isolated Kupffer cells from 2-AAF/PH rat liver showed high activity of secreting TNF-a and IL-6 at six hours after PH. Depletion of Kupffer cells did not affect response of hepatic stellate cells, as kinetics of hepatic stellate cell activation was found to be accompanied by the proliferation of oval cells to a similar extent in both group, and there was also no significant difference of HGF expression between the two groups at all the time points after PH.Conclusions:Kupffer cells play an important role on oval cell expansion in rat 2-AAF/PH model, most likely via secreting important growth-stimulating factors, such as TNF-αand IL-6, and then phosphorylating STAT3Objectives:In order to evaluate the role of Kupffer cells in a rat model of 2-AAF/PH-induced liver injury.Methods:All rats received treatment of 2-AAF/PH, and then randomly divided into Kupffer cell depleted group and non-depleted group. Accumulated survival rate between the two groups was measured. Blood serum was collected for analysis of alanine transaminase (ALT) and aspartate aminotransferase (AST) activity. Liver damage was determined in H&E staining liver sections. Immunohistochemistry for caspase-3 as well as TUNEL staining were employed to assess hepatocytes apoptosis. ELISA was performed for IL-10 concentration in liver homogenate.Results:All of the 2-AAF/PH rats treated with saline remained alive throughout the time period studied. However, Kupffer cell depleted rats became weak after PH and a significantly unexpected mortality rate was observed. Few rats in this group remained alive beyond 9 days (P<0.01). There were obvious hepatocyte degeneration and necrosis foci in depleted group, while no obvious pathological change was observed in control rat livers. Moreover, Number of apoptotic hepatocytes in Kupffer cell depleted rats was significantly much higher than that in control rats 24 hours after PH (P<0.01). In addition, Kupffer cell depletion resulted in significant decrease of IL-10 concentration in liver.Conclusions:Kupffer cells reveal a protective role in 2-AAF/PH-induced liver injury partly through producing anti-inflammatory cytokine IL-10.